Alzheimer's disease (AD) is pathologically characterized by the abundance of extracellular deposition of amyloid β-peptide (Aβ) as amyloid plaques and vascular amyloid, and the intraneuronal accumulation of neurofibrillary tangles (Selkoe, 2001). AD therapy is currently not essential. There are few drugs available, and most compounds for AD are acetylcholine esterase inhibitors, which aim at stabilizing acetylcholine levels in the synaptic cleft to maintain neurotransmission (Cacabelos et al., 2000). Many researchers favor other therapeutic approaches that target formation, deposition, and clearance of Aβ from nervous tissue. Vaccination (Schenk et al., 1999; Janus et al., 2000; Morgan et al., 2000) and secretase inhibitors (Skovronsky et al., 2000) have been reported as experimental therapies and for clinical trials.

Based on a nucleation-dependent polymerization model to explain the mechanism of Alzheimer's β-amyloid fibril (fAβ) formation in vitro (Jarrett and Lansbury, 1993; Lomakin et al., 1997; Naiki et al., 1997; Naiki and Gejyo, 1999), we reported previously that nordihydroguaiaretic acid (NDGA) and rifampicin (RIF) dose-dependently inhibit fAβ formation from Aβ and fAβ extension in vitro (Naiki et al., 1998). In addition, we reported that NDGA also destabilizes fAβ(1–40) and fAβ(1–42) in a concentration-dependent manner within a few hours at pH 7.5 at 37°C, based on fluorescence spectroscopic analysis with thioflavin T (ThT) and electron microscopic studies (Ono et al., 2002b). Very recently, we also showed that wine-related polyphenols, such as myricetin, dose-dependently inhibit formation and extension of fAβ and destabilize preformed fAβs in vitro (Ono et al., 2003).

Many studies have demonstrated that oxidative damage is closely associated with the hallmark pathologies of AD and may play a critical role in the development of AD (Pratico and Delanty, 2000; Rottkamp et al., 2000; Smith et al., 2000; Varadarajan et al., 2000). Oxidative stress in AD may result from aging, energy deficiency, inflammation, or excessive production of Aβ (Grundman and Delaney, 2002). Many antioxidant compounds, such as vitamin E (DL-α-tocopherol) (Behl et al., 1992; Zhou et al., 1996; Subramaniam et al., 1998; Pereira et al., 1999; Yatin et al., 1999), vitamin A (Jama et al., 1996; Perrig et al., 1997), quercetin (Roth et al., 1999), and nicotine (Kihara et al., 1997) have been suggested to reduce oxidative stress associated with AD. One phenolic antioxidant alternative is curcumin (Cur), a major component of the yellow curry spice turmeric. This spice is used in the traditional diet and as an herbal medicine in India (Kelloff et al., 1996, 2000). The frequency of AD in India is roughly one-quarter of that in the US (e.g., 0.7 vs. 3.1% in patients between 70 and 79 yr; Ganguli et al., 2000). Cur is much stronger than vitamin E as a free radical scavenger (Zhao et al., 1989), protects the brain from lipid peroxidation (Martin-Aragon et al., 1997), and scavenges nitric oxide (NO)-based radicals (Sreejayan and Rao, 1997). Oral administration of Cur has been shown to be centrally neuroprotective (Kaul and Krishnakantha, 1997; Rajakrishnan et al., 1999). Recently, Lim et al. (2001) reported that Cur reduced oxidative damage and amyloid pathology in an Alzheimer transgenic amyloid precursor protein with Swedish mutant (APPSw) mouse model (Tg2576). Although Lim et al. (2001) suggested that Cur blocks AD pathogenesis at multiple sites in the inflammation cascade, the direct effects of Cur on the formation and destabilization of fAβ remain unclear.

Using fluorescence spectroscopy with ThT and electron microscopy, we examined the effects of Cur and its analog, rosmarinic acid (RA), on the formation and extension of fAβ(1–40) and fAβ(1–42), as well as their activity to destabilize fAβs at pH 7.5 at 37°C in vitro.

MATERIALS AND METHODS

Preparation of Aβ and fAβ Solutions

Aβ(1–40) (trifluoroacetate salt, 520130; Peptide Institute, Osaka, Japan) and Aβ(1–42) (trifluoroacetate salt, 520625; Peptide Institute) were dissolved by brief vortexing in 0.02% ammonia solution at a concentration of 500 μM (2.2 mg/mL) and 250 μM, respectively, in a 4°C room and then stored at −80°C before assaying (fresh Aβ[1–40] and Aβ[1–42] solutions). fAβ(1–40) and fAβ(1–42) were formed from the fresh Aβ(1–40) and Aβ(1–42) solutions, respectively, sonicated, and then stored at 4°C as described elsewhere (Hasegawa et al., 1999).

Fresh, nonaggregated fAβ(1–40) and fAβ(1–42) was obtained by extending sonicated fAβ(1–40) or fAβ(1–42) with fresh Aβ(1–40) or Aβ(1–42) solutions, respectively, just before the destabilization reaction (Ono et al., 2002a, b). The reaction mixture volume was 600 μL and contained 10 μg/mL (2.3 μM) fAβ(1–40) or fAβ(1–42), 50 μM Aβ(1–40) or Aβ(1–42), 50 mM phosphate buffer, pH 7.5, and 100 mM NaCl. Measurement of ThT fluorescence showed that the extension reaction proceeded to equilibrium after incubation at 37°C for 3–6 hr under nonagitated conditions. In the following experiment, the concentration of fAβ(1–40) and fAβ(1–42) in the final reaction mixture was regarded as 50 μM.

A fluorescence spectroscopic study was carried out as described elsewhere (Naiki and Nakakuki, 1996) on a Hitachi F-2500 fluorescence spectrophotometer. Optimum fluorescence measurements of fAβ(1–40) and fAβ(1–42) were obtained at the excitation and emission wavelengths of 445 and 490 nm, respectively, with the reaction mixture containing 5 μM ThT (Wako Pure Chemical Industries, Osaka, Japan) and 50 mM of glycine-NaOH buffer, pH 8.5. An electron microscopic study and polarized light microscopic study of the reaction mixtures were carried out as described elsewhere (Hasegawa et al., 1999).

Aliquots (30 μL) of the mixture were put into oil-free PCR tubes (size, 0.5 mL, code number 9046; Takara Shuzo, Otsu, Japan) and the tubes were then put into a DNA thermal cycler (PJ480; Perkin Elmer Cetus, Emeryville, CA). Starting at 4°C, the plate temperature was elevated at maximal speed to 37°C. Incubation times ranged between 0–8 days (as indicated in each figure), and the reaction was stopped by placing the tubes on ice. The tubes were not agitated during the reaction. Aliquots (5 μL) from each tube in triplicate were subjected to fluorescence spectroscopy and the mean of the three measurements determined. In the ThT solution, the concentration of Cur, RA, and NDGA examined in this study was diluted up to 1/200 of that in the reaction mixture. We confirmed that these compounds did not quench ThT fluorescence at the diluted concentration (data not shown).

After being mixed by pipetting, triplicate 5-μL aliquots of the reaction mixture were subjected to fluorescence spectroscopy and 30-μL aliquots were put into PCR tubes. Reaction tubes were then transferred into a DNA thermal cycler. Starting at 4°C, the plate temperature was elevated at maximal speed to 37°C. Incubation times ranged from 0–6 hr (as indicated in each figure), and the reaction was stopped by placing the tubes on ice. The reaction tubes were not agitated during the reaction. Aliquots (5 μL) from each tube in triplicate were subjected to fluorescence spectroscopy and the mean of the three measurements was determined.

Other Analytical Procedures

Protein concentrations of the reaction mixture supernatants after centrifugation were determined by the method of Bradford (1976) with a protein assay kit (Bio-Rad, Hercules, CA). The Aβ(1–40) solution quantified by amino acid analysis was used as the standard. Linear least squares fit was used for statistical analysis. The effective concentrations (EC50) were defined as the concentrations of NDGA, Cur, or RA required to inhibit the formation or extension of fAβs to 50% of the control value, or the concentrations to destabilize fAβs to 50% of the control value. EC50 were calculated by the sigmoidal curve fitting of the data using Igor Pro v.4 (WaveMetrics, Lake Oswego, OR).

RESULTS

Effects of Cur and RA on Kinetics of fAβ Formation

As shown in Figure 1A–D, when fresh Aβ(1–40) or Aβ(1–42) was incubated at 37°C, the fluorescence of ThT followed a characteristic sigmoidal curve. This curve is consistent with the nucleation-dependent polymerization model (Jarrett and Lansbury, 1993; Naiki et al., 1997). fAβ(1–40) and fAβ(1–42) stained with Congo red showed typical orange-green birefringence under polarized light (data not shown). The final equilibrium level decreased after incubation of Aβ(1–40) or Aβ(1–42) with 10 and 50 μM Cur or RA (Fig. 1A–D).

As shown in Figure 2A–D, when fresh Aβ(1–40) or Aβ(1–42) was incubated with fAβ(1–40) or fAβ(1–42), respectively, at 37°C, the fluorescence increased hyperbolically without a lag phase and proceeded to equilibrium much more rapidly than without seeds (compare Fig. 1 and 2). This curve is consistent with a first-order kinetic model (Naiki and Nakakuki, 1996). When Aβ(1–40) and fAβ(1–40) were incubated with 10 or 50 μM Cur or RA, the final equilibrium level decreased (Fig. 2A,C). A similar effect of Cur and RA was observed for the extension of fAβ(1–42) (Fig. 2B,D). At a constant fAβ(1–40) concentration, a perfect linearity was observed between Aβ(1–40) concentration and the initial rate of fAβ(1–40) extension in both the presence and absence of Cur (Fig. 2E). This linearity is again consistent with a first-order kinetic model and indicates that at each Aβ(1–40) concentration, the net rate of fAβ(1–40) extension is the sum of the rates of polymerization and depolymerization (Naiki and Nakakuki, 1996, Hasegawa et al., 2002). In the presence of 10 μM Cur, the slope of the straight line decreased to about 1/2. The interpretation of this figure, implicating the mechanism of the anti-amyloidogenic effect of Cur, will be discussed later.

Fibril Destabilizing Assay

As shown in Figure 4A–D, the fluorescence of ThT was almost unchanged during the incubation of fresh fAβ(1–40) or fAβ(1–42) at 37°C without additional molecules. On the other hand, the ThT fluorescence decreased immediately after addition of Cur and RA to the reaction mixture. After incubation of 25 μM fresh fAβ(1–40) with 50 μM Cur for 1 hr, many short, sheared fibrils were observed (Fig. 5B). At 4 hr, the number of fibrils was reduced markedly, and small amorphous aggregates were occasionally observed (Fig. 5C). Similar morphology was observed after incubation of 25 μM fresh fAβ(1–42) with 50 μM Cur (data not shown). RA also destabilized preformed fAβ(1–40) and fAβ(1–42) similarly (data not shown).

After incubation with 50 μM Cur or RA for 4 hr, fAβ(1–40) and fAβ(1–42) were not stained with Congo red as much as fresh fAβ(1–40) and fAβ(1–42) were (data not shown). They all showed orange-green birefringence under polarized light (data not shown), however, indicating that a significant amount of intact fAβ(1–40) and fAβ(1–42) remained in the mixture after the reaction. When the protein concentration of the supernatant after centrifugation (at 16,000 × g for 2 hr at 4°C) was measured by the Bradford assay, no proteins were detected in the supernatant in any case (data not shown). This implied that although Cur and RA could destabilize fAβ(1–40) and fAβ(1–42) to visible aggregates (Fig. 5C), they could not depolymerize fAβ(1–40) and fAβ(1–42) to monomers or oligomers of Aβ(1–40) and Aβ(1–42).

Comparison of NDGA, Cur, and RA Activity

NDGA, Cur and RA dose-dependently inhibited fAβ formation and extension and destabilized preformed fAβs. We calculated EC50, the concentrations of NDGA, Cur, or RA to inhibit the formation or extension of fAβs to 50% of the control value or to destabilize fAβs to 50% of the control value, by the sigmoidal curve fitting of the data as shown in Figures 1E, 2F, and 4E (Table I). In all molecules examined, EC50 values required to inhibit fAβ formation or extension were similar to those required to destabilize fAβs. All data presented in Table I suggest that NDGA, Cur, and RA have similar anti-amyloidogenic activity.

Table I. EC50 of NDGA, Cur, and RA for Formation, Extension, and Destabilization of fAβ(1–40) and fAβ(1–42)*

EC50 defined as the concentrations of nordihydroguaiaretic acid (NDGA), curcumin (Cur), or rosmarinic acid (RA) to inhibit the formation or extension of fAβs to 50% of the control value or the concentrations to destabilize fAβs to 50% of the control value. EC50 was calculated by sigmoidal curve fitting of the data.

Figure 6.

Cur, RA, and NDGA did not extend the length of the lag phase in the formation fAβs from Aβs (Fig. 1; Naiki et al., 1998). In addition, they did not extend the time to proceed to equilibrium in the extension reaction (Fig. 2; Naiki et al., 1998). These results are in sharp contrast to those of apolipoprotein E (apoE), in which apoE extends in a dose-dependent manner the length of lag phase and the time to proceed to equilibrium (Naiki et al., 1998). Although apoE was suggested to inhibit the formation of fAβs in vitro by making a complex with Aβs, thus eliminating free Aβs from the reaction mixture (Naiki et al., 1997, 1998), Cur, RA, and NDGA may inhibit the formation of fAβs by different mechanisms. As shown in Figure 2E, the extension of fAβ(1–40) followed a first-order kinetic model even in the presence of Cur. The net rate of fAβ(1–40) extension is the sum of the rates of polymerization and depolymerization (Naiki and Nakakuki, 1996, Hasegawa et al., 2002). One possible explanation for the finding in Figure 2E thus may be that Cur could bind to the ends of extending fAβ(1–40) and increase the rate of depolymerization by destabilizing the conformation of Aβ(1–40) that has just been incorporated into the fibril ends. Alternatively, Cur would bind to Aβ(1–40) and consequently decrease the rate of polymerization. Further studies are essential to clarify the mechanisms by which Cur and RA inhibit fAβ formation in vitro.

Cur is a potent antioxidant and an effective antiinflammatory compound (Zhao et al., 1989; Sreejayan and Rao, 1997; Xu et al., 1998). Part of its nonsteroidal antiinflammatory drug-like activity is based on the inhibition of nuclear factor κB (NFκB)-mediated transcription of inflammatory cytokines (Xu et al., 1998), inducible nitric oxide synthase (iNOS) (Chan et al., 1998), and cyclooxygenase 2 (Cox-2) (Zhang et al., 1999). Because of antitumor activity, relative safety, and its long history of use, Cur is currently being developed for clinical use as a cancer chemopreventive agent in India (Kelloff et al., 1996, 2000). RA is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid (Petersen and Simmonds, 2003). It is commonly found in species of the Boraginaceae and the subfamily Nepetoideae of the Lamiaceae (Petersen and Simmonds, 2003), and has several interesting biological activities, e.g., antioxidant, antiinflammatory, antimutagen, antibacterial, and antiviral (Parnham and Kesselring, 1985). The latter activity is used in the therapy of Herpes simplex virus infections with RA-containing extracts of Melissa officinalis. It is thought that the antiinflammatory properties are based on the inhibition of lipoxygenases and cyclooxygenases, as well as the interference with the complement cascade (Parnham and Kesselring, 1985). RA is eliminated rapidly from the blood circulation after intravenous administration and shows very low toxicity in mice (Parnham and Kesselring, 1985). NDGA is a pure compound isolated from the creosote bush, Larrea tridentata (Luo et al., 1998). It is a potent oxygen radical scavenger and a lipoxygenase inhibitor (Goodman et al., 1994), and has been capable of lowering plasma glucose concentration in mouse models of non-insulin-dependent diabetes mellitus (Luo et al., 1998). Our study and several other reports suggest that Cur, RA, and NDGA could be key molecules for development of therapeutics for AD. First, Kim et al. (2001) reported that Cur protects PC12 and human umbilical vein endothelial cells from Aβ insult by strong antioxidant properties. Goodman et al. (1994) also reported that NDGA can interrupt the cytotoxicity of Aβ to cultured rat hippocampal neurons by suppressing Aβ-induced accumulation of reactive oxygen species and intracellular free Ca2+. Second, Lim et al. (2001) reported that Cur suppresses indices of inflammation and oxidative damage in the brain of Tg2576 APPSw transgenic mouse and that low, nontoxic doses of Cur decrease levels of insoluble and soluble Aβ and plaque burden in many affected brain regions. They speculated that mechanisms are based mainly on inflammation-related targets, such as inhibition of NFκB-induced iNOS, Cox-2, and inflammatory cytokine production. Finally, we showed that Cur, RA, and NDGA dose-dependently inhibit fAβ formation from fresh Aβ and destabilize preformed fAβ in vitro. In addition, cell culture experiments with human embryonic kidney (HEK) 293 cells indicated that fAβ destabilized by NDGA might be less toxic than are intact fAβ (Ono et al., 2002b). It thus may be reasonable to speculate that Cur, RA, and NDGA could prevent the development of AD not only through scavenging reactive oxygen species but also through directly inhibiting fAβ deposition in the brain. Recently, Caughey et al. (2003) showed that Cur potently inhibits protease-resistant prion protein accumulation in scrapie agent-infected neuroblastoma cells. Although the exact mechanism of anti-amyloidogenic activity of Cur, RA, and NDGA is unclear, these structurally similar compounds could be key molecules for the development of therapeutics for AD and other human conformational diseases.

Acknowledgements

This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grant-in-Aid for Scientific Research to M.Y. and a Grant-in-Aid for Scientific Research on Priority Areas [C] -Advanced Brain Science Project to H.N.) and by the Ministry of Health, Labour, and Welfare, Japan (grant to M.Y.). We thank Drs. S. Okino and K. Iwasa (Kanazawa University) for cooperation in the experiments, and Drs. H. Okada and N. Takimoto (Fukui Medical University) for excellent technical assistance.